Nucleophilic oxime reactivators of organophosphate (OP) inhibited human acetylcholinesterase (hAChE) are reactivating antidotes against OP intoxication from nerve agent (e.g., VX, sarin) or OP pesticide (e.g., paraoxon) exposure. Within the past decade it has become increasingly clear that for effective and complete recovery from OP intoxication antidotal action is needed in both peripheral and central nervous system tissues. We are using structure-based design to create uncharged bis-oxime reactivators that possess superior in vitro properties for reactivation of various OP-conjugates of hAChE compared to the known charged pyridinium aldoxime-based therapies. Our strategies involve studying hAChE from the atomic to the molecular level by employing a combination of experimental and theoretical techniques, including X-ray and neutron crystallography, neutron vibrational spectroscopy and molecular simulations. Starting with RS-170B, MMB4 (both charged) and RS194B (uncharged) oximes we have obtained a clear view of their binding to native and VX- or paraoxon-inhibited hAChE. Neutron vibrational spectra in the 5-50 cm-1 frequency regime indicated softening of the vibrational dynamics in the paraoxon-bound hAChE, pointing to significant softening of picosecond vibrational motions of the acyl pocket loop that must accommodate one of the pesticide's ethoxy groups. We have used our crystal structures to design and test uncharged bis-oximes, demonstrating in vitro that several of them exceeded efficacy of RS194B in reactivation of sarin, cyclosarin, VX and paraoxon inhibited hAChE. To further the reactivator design, we are working on obtaining neutron structures of hAChE which will provide direct experimental observation of hydrogen atoms to determine protonation states of hAChE residues and bound reactivators. Our designed uncharged bis-oxime antidotes are a conceptually novel, effective scaffold of nucleophilic reactivators and a promising new resource for creation of adjustable, accelerated centrally active antidotes against OP intoxication.
Acetylcholinesterase (AChE ; EC 3.1.1.7), is a primary target in acute intoxication by nerve agents and organophosphate (OP) pesticides that covalently inhibit its remarkably high catalytic activity. The high worldwide incidence of OP poisoning causing more than 200, 000 fatalities annually obviates a need for more effective therapies of OP intoxication. Our approach is to develop more efficient oxime reactivators of both AChE and butyrylcholinesterase (BChE ; EC 3.1.1.8) inhibited by OPs. Design of novel, accelerated oxime reactivators of OP-conjugated tissue AChE yields reactivation acceleration achieved by improved understanding of human AChE (hAChE) structure at the room temperature and in aqueous solution guided by advanced biophysical studies and computational molecular dynamics simulations. Here, we demonstrate previously undetected changes in hAChE solution structure, remote from the active center, upon OP inhibition and oxime reactivation by using solution small-angle X-ray scattering profiles of hAChE. We have also resolved first room temperature (22 oC) X-ray crystal structures of hAChE in complex with oximes and other ligands, that reflect their physiological interactions more accurately than X-ray structures conventionally determined at -173 oC (100 K). Additionally, we focus on catalytic detoxification of offending OP toxicants in exposed tissue by oxime assisted OP bioscavengers before these inhibit tissue AChE. We developed both BChE based and mutant hAChE based, oxime assisted, OP bioscavengers and determined their X-ray structure in order to rationally improve their detoxification efficacy. The advanced hAChE mutant-based and BChE-based bioscavenging, have demonstrated fast in vitro and ex vivo OP detoxification and improvement in survival of nerve agent OP-exposed mice upon treatment with bioscavengers, even in exposure to the fastest-aging nerve agent soman. Allosteric enhancement of the BChE-based oxime assisted OP bioscavenging, accelerated by small molecule allosteric modulators yielded in our hands impressive in vivo improvements of oxime antidotal therapies of OP exposed mice. In conclusion, we provide here the evidence that tertiary structures of both AChE and BChE macromolecules posses’ intrinsic potential for allosteric regulation of significant importance for structure-based developed therapies of OP intoxication. Supported by the NIH CounterACT Program, the NIH Office of the Director, and the National Institute of Neurological Disorders and Stroke, grant numbers 1U01NS083451, R21NS072086 and 1R21NS084904.
Xylanases catalyze the hydrolysis of plant hemicellulose xylan into oligosaccharides by cleaving the main-chain glycosidic linkages connecting xylose subunits. To study ligand binding and to understand how the pH constrains the activity of the enzyme, variants of the Trichoderma reesei xylanase were designed to either abolish its activity (E177Q) or to change its pH optimum (N44H). An E177Q–xylohexaose complex structure was obtained at 1.15 Å resolution which represents a pseudo-Michaelis complex and confirmed the conformational movement of the thumb region owing to ligand binding. Co-crystallization of N44H with xylohexaose resulted in a hydrolyzed xylotriose bound in the active site. Co-crystallization of the wild-type enzyme with xylopentaose trapped an aglycone xylotriose and a transglycosylated glycone product. Replacing amino acids near Glu177 decreased the xylanase activity but increased the relative activity at alkaline pH. The substrate distortion in the E177Q–xylohexaose structure expands the possible conformational itinerary of this xylose ring during the enzyme-catalyzed xylan-hydrolysis reaction.
Room-temperature and 100 K X-ray and room-temperature neutron diffraction data have been measured from equine cyanomethemoglobin to 1.7 Å resolution using a home source, to 1.6 Å resolution on NE-CAT at the Advanced Photon Source and to 2.0 Å resolution on the PCS at Los Alamos Neutron Science Center, respectively. The cyanomethemoglobin is in the R state and preliminary room-temperature electron and neutron scattering density maps clearly show the protonation states of potential Bohr groups. Interestingly, a water molecule that is in the vicinity of the heme group and coordinated to the distal histidine appears to be expelled from this site in the low-temperature structure.
HIV-1 protease inhibitors are crucial for treatment of HIV-1/AIDS, but their effectiveness is thwarted by rapid emergence of drug resistance. To better understand binding of clinical inhibitors to resistant HIV-1 protease, we used room-temperature joint X-ray/neutron (XN) crystallography to obtain an atomic-resolution structure of the protease triple mutant (V32I/I47V/V82I) in complex with amprenavir. The XN structure reveals a D+ ion located midway between the inner Oδ1 oxygen atoms of the catalytic aspartic acid residues. Comparison of the current XN structure with our previous XN structure of the wild-type HIV-1 protease-amprenavir complex suggests that the three mutations do not significantly alter the drug–enzyme interactions. This is in contrast to the observations in previous 100 K X-ray structures of these complexes that indicated loss of interactions by the drug with the triple mutant protease. These findings, thus, uncover limitations of structural analysis of drug binding using X-ray structures obtained at 100 K.
